Volume 14, Issue 5, Pages 979-990 (February 2016) Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities  Jorge Franco, Uthra Balaji, Elizaveta Freinkman, Agnieszka K. Witkiewicz, Erik S. Knudsen  Cell Reports  Volume 14, Issue 5, Pages 979-990 (February 2016) DOI: 10.1016/j.celrep.2015.12.094 Copyright © 2016 The Authors Terms and Conditions

Cell Reports 2016 14, 979-990DOI: (10.1016/j.celrep.2015.12.094) Copyright © 2016 The Authors Terms and Conditions

Figure 1 CDK4/6 Inhibition Leads to Mitochondria Accumulation in an RB-Dependent Fashion (A) Representative flow cytometry histogram displaying cellular complexity on the x axis for cells treated with PD-0332991 for 120 hr. (B) Relative ATP levels from cells treated with control or PD-0332991 for 120 hr. The average and SD are shown, and the statistical comparison to vehicle control was determined by t test (∗∗p < 0.01, ∗∗∗p < 0.001). (C) Confocal fluorescence imaging of mitochondria (62.5×) in the indicated cells treated with PD-0332991 for 120 hr (scale bar, 20 μm). (D) Flow cytometry histograms showing comparison of MitoTracker red fluorescence of the control versus PD-0332991-treated cells. (E) Representative transmission electron microscope micrograph of control and PD-0332991-treated PL5 cells (scale bar, 1 μm). Quantification of mitochondria from counting high-power fields and the average and SD are shown. The statistical comparison to vehicle control was determined by t test (∗∗∗p < 0.001). (F) Flow cytometry histograms showing comparison of MitoTracker red fluorescence of the control versus PD-0332991-treated cells following CCND1 or RB knockdown. (G) Quantitation of MitoTracker red intensity following treatment with PD-0332991 or transduction of the constitutively active RB allele 7-LP. The average and SD are shown. The statistical comparison to vehicle control was determined by t test (∗p < 0.05, ∗∗p < 0.01). (H) Flow cytometry histograms showing comparison of MitoTracker red fluorescence of the control versus PD-0332991-treated cells in the EMC43 cell model. (I) H&E staining, immunohistochemistry for (Ki67) (scale bar, 100 μm), and fluorescence (scale bar, 200 μm) imaging (TOM20/Vimentin/DAPI) of patient-derived xenograft EMC43. See also Figure S1. Cell Reports 2016 14, 979-990DOI: (10.1016/j.celrep.2015.12.094) Copyright © 2016 The Authors Terms and Conditions

Figure 2 CDK4/6 Inhibition Leads to Metabolic Reprogramming (A) Oxygen consumption rate (OCR) of PL45 and Capan2 cell lines either control or treated with PD-0332991. (B) Quantification of media glucose and glutamine uptake, and glutamate and lactate production from the indicated cell lines with or without PD-0332991 treatment. The average and SD are shown. The statistical comparison to vehicle control was determined by t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (C) Extracellular acidification rate (ECAR) in cells treated with or without PD-0332991. (D) Relative fold change of glycolytic intermediates Glucose 6P; Fructose 1,6BP; pyruvate; and lactate as determined by mass spectrometry. The average and SD are shown. The statistical comparison to vehicle control was determined by t test. (E) Flux analysis of cell populations treated with universally labeled 13C-glucose (solid bars) or universally labeled 13C-glutamine (stippled bars). Bars show the relative average abundance and SD of differing mass species. (F) Relative fold change of TCA intermediates alpha-ketoglutare, citrate, malate, and fumarate. The average and SD are shown. The statistical comparison to vehicle control was determined by t test (∗∗p < 0.01). (G) Flux analysis of cell populations treated with universally labeled 13C-glucose (solid bars) or universally labeled 13C-glutamine (stippled bars). Bars show the relative average abundance and SD of differing mass species. (H) Quantification of cell viability following acute glucose or glutamine withdrawal. The average and SD are shown. The statistical comparison to vehicle control was determined by t test (∗p < 0.05). (I) Representative images of crystal violet stained cells following either glutamine or glucose withdrawal (scale bar, 200 μm). See also Figure S2. Cell Reports 2016 14, 979-990DOI: (10.1016/j.celrep.2015.12.094) Copyright © 2016 The Authors Terms and Conditions

Figure 3 CDK4/6 Inhibition Leads to the Induction of MTOR Activity and Downstream Effects on Metabolism (A) Quantification of reverse phase protein array (RPPA) data. The log-fold change of proteins abundance with PD-0332991 treatment is displayed as a function of the 217 protein species detected on the array. RPPA data for selected proteins and phosphoproteins is shown, with the average abundance and SD. The statistical comparison to vehicle control was determined by t test (∗p < 0.05, ∗∗p < 0.01). (B) Immunoblotting was performed for the indicated proteins in cells treated with the indicated pathway selective inhibitors. (C) Immunohistochemical analysis of xenograft tumors treated with lactate control or PD-0332991 for 8 days. Representative images of Ki67 and pS6 (S235/236) are shown (scale bar, 100 μm). (D) Gene expression profiling was performed on Capan 2 cells treated with control or PD-0332991 for 120 hr. The heatmap shows genes passing a 1.5-fold p < 0.05 cutoff. Representative genes falling into selected gene set enrichment categories are shown in the heatmap. (E) Quantification of BrdU positive cells following 24 hr treatment with dual PI3K/MTOR (BEZ235), PI3K (BKM120, BYL719), mTOR (Torin, Everolimus, and Deforolimus), and MEK (AZD-6244) inhibitors alone and in combination with PD-0332991 treatment. The average BrdU incorporation and SD are shown. The statistical comparison to single agent treatment was determined by t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (F) Bright-field images of PD-0332991, AZD-6244, and Torin 1 and combination treated cells (scale bar, 100 μm). (G–J) Quantification of mitochondria (G), OCR (H), ECAR (I), and ATP (J) levels from cells treated with the indicated drugs and combinations. The average signals and SD are shown. The statistical comparison to control was determined by t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S3. Cell Reports 2016 14, 979-990DOI: (10.1016/j.celrep.2015.12.094) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Selective Cooperation with CDK4/6 Inhibition for Enhancing Therapeutic Effect (A) Quantification of SA-β-gal positive cells following the indicated treatments. The average number of senescent cells and SD are shown. The statistical comparison to control was determined by t test (∗∗p < 0.01, ∗∗∗p < 0.001). (B) Quantification of apoptotic cells following the indicated treatments. The average number of apoptotic cells and SD are shown. The statistical comparison to control was determined by t test (∗∗∗p < 0.001). (C) Colony formation was assessed at the indicated times by crystal violet staining. Representative images are shown. (D) Tumor volume of PL5 xenografts following treatment with BEZ235, PD-0332991, and the combination was measured as a function of time. The average volume and SD are plotted, and the statistical comparison versus vehicle treated control is shown by t test (∗p < 0.05, ∗∗p < 0.01). (E) Representative Ki-67 immunohistochemistry of the PL5 treated xenografts (scale bar, 100 μm). (F) Tumor volume of orthotopically implanted PL45 cells that were treated with the indicated agents. The average volume, SD, and statistical comparison to the vehicle treated control are shown. (G) Representative histograms showing fluorescence of total ROS in the presence versus absence of PD-0332991. (H) Surviving cells following HO-1 or CAT knockdown in the absence or presence of PD-0332991 was determined. Bars show the average surviving fraction relative to transfected controls. The statistical analysis to the RNAi control was determined by t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (I) Colony outgrowth of knockdown cells alone or in combination with PD-0332991. Representative images are shown. (J) Fold change in fraction of apoptotic cells pretreated with PD-0332991 for the indicated period of time, followed by acute treatment with ABT-737. The average number of apoptotic cells and standard SD are shown. The statistical analysis to single agent versus combination with PD-0332991 was determined by t test (∗p < 0.05). (K) Impact of PD-0332991 pretreatment on sensitivity to ABT-737. The average and SD are shown. The statistical comparison to control was determined by t test (∗p < 0.05, ∗∗p < 0.01). (L) Prolonged treatment with ABT-737 of control or PD-0332991 pretreated and stained for crystal violet. See also Figure S4. Cell Reports 2016 14, 979-990DOI: (10.1016/j.celrep.2015.12.094) Copyright © 2016 The Authors Terms and Conditions